October 7, 2020

Current standard

Weissman/Ingolia method

  • Used in Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

  • Successful in 9/20 attempts (all 8 samples in HSA expression study had rRNA contamination)

Updated method

1: Prepare a cell lysate

2: Nuclease footprinting and ribosome recovery

3: Footprint fragment purification

4: Dephosporylation and polyadenylation

5: Reverse transcription with TSO

New England Biolabs (n.d.)

5: Reverse transcription with TSO

5: Inherent drawback: concatenation

6: rRNA reduction with double stranded nuclease

Chung et al. (2015)

7: PCR Amplifcation

8: Next generation sequencing

References